Recently, animal cell culture techniques have been remarkably developed, and also research and development related to animal cells have been carried out in a wide variety of fields. The animal cells have been used not only for commercializing them at the early stage of development or for commercializing products from the cells, but also for analyzing cells and their surface proteins to design useful medicines or for performing treatment by growing the cells of patients in vitro or enhancing the function of the cells, followed by returning the cells into the bodies of the patients. At present, many researchers pay attention to the techniques for culturing animal cells.
Many animal cells including human cells are anchorage-dependent cells. Therefore, in order to culture animal cells in vitro, the cells need to be once attached to a scaffold substrate. Under such a background, many researchers have previously designed and devised substrate surfaces that are more desirable for cells. However, all these techniques are involved in those during cell culture. Cultured anchorage-dependent cells produce adhesive proteins when they adhere to a substrate. Therefore, in conventional techniques, the adhesive proteins need to be destroyed, e.g., by enzymatic treatment or the like for detaching the cells. Consequently, these techniques have a drawback in that cell surface proteins, which are specific for each cell and are produced from the cells during culturing them, are also simultaneously destroyed. However, any countermeasures against this problem have not been found or investigated.
Under such a background, Japanese Laid-open Patent Publication No. 2-211865 (Patent Literature 1) describes a novel method of culturing cells, comprising the step of culturing cells on a cell culture support coated with a polymer whose upper or lower critical solution temperature in water is 0 to 80° C., at the upper critical solution temperature or less, or the lower critical solution temperature or more, and then detaching the cultured cells without enzymatic treatment by increasing the culturing temperature to the upper critical solution temperature or more, or decreasing the culturing temperature to the lower critical solution temperature or less. Japanese Laid-open Patent Publication No. 05-192138 (Patent Literature 2) describes that skin cells are detached with low damage from a cell culture substrate, by culturing skin cells on the temperature-responsive cell culture substrate described in Japanese Laid-open Patent Publication No. 2-211865 (Patent Literature 1), at the upper critical solution temperature or less, or the lower critical solution temperature or more, and then increasing the culturing temperature to the upper critical solution temperature or more, or decreasing the culturing temperature to the lower critical solution temperature or less. In addition, Japanese Patent Application No. 2007-105311 (Patent Literature 3) describes a method of repairing the surface protein of the cultured cells using the temperature-responsive cell culture substrate described in Japanese Laid-open Patent Publication No. 2-211865 (Patent Literature 1). Such use of the temperature-responsive cell culture substrate has allowed various developments of conventional culture techniques.
However, the conventional techniques have problems in that the surface of a chemically inert engineering plastic must be coated with a polymer by using high energy such as electron beams, a large-scale apparatus such as an electron beam irradiation apparatus is needed for coating the surface, and thus the cell culture substrate inevitably becomes expensive.
In order to solve such problems, some techniques have been previously developed. As described in, e.g., Soft Matter, 5, 2937-2946 (2009) (Non Patent Literature 1) or Interface, 4, 1151-1157 (2007) (Non Patent Literature 2), a method of coating a substrate surface with a polymer having a particular molecular structure is known. However, all the techniques do not reach the technical level in which cells can be cultured, similarly to a case where the conventional cell culture substrates are used, and the level in which the cells can be detached only by changing temperature, similarly to a case where the temperature-responsive cell culture substrate which is produced by using the above-described electron beams, or the level in which cultured cells can be detached as a cell sheet when they become confluent.